Qualitative and quantitative changes in sialomucins during 1,2-dimethylhydrazine-induced colon carcinogenesis in the rat.

Regional differences in goblet cell glycoproteins have been demonstrated qualitatively and, to a limited extent, quantitatively in the normal adult colon. In disease states, alterations in these glycoproteins, particularly the sialoglycoproteins (SGs), have been reported. The present study defined parallel qualitative and quantitative changes in SGs in three colon regions during 1,2-dimethylhydrazine [(DMH) CAS: 540-73-8]-induced carcinogenesis. SGs were assessed histochemically by use of high iron diamine-Alcian blue (pH 2.5) staining, and tissue sialic acid levels were measured by a modified Warren assay. Two groups of inbred SD rats (n = 28) were pair-fed nutritionally complete liquid diets with 36% of calories supplied as ethanol or isocaloric carbohydrates. The dietary alcohol was added to selectively enhance rectal tumors, a region of prevalent tumors in humans. Both groups received 4 weeks of liquid diet followed by 4 weeks of standard laboratory chow with weekly sc injections of DMH. This 8-week cycle was repeated four times (32 wk). Animals from each group were sacrificed at 8, 16, 24, and 32 weeks, and adjacent tissues from proximal and distal colon and rectum were prepared for histology and biochemical assay. The results showed a progressive increase in sialomucin staining in normal-appearing mucosa in distal colon and rectum in both groups but not in the proximal colon. In contrast, tissue sialic acid increased in all three regions as early as 8 weeks, and significant increases were consistently present by 32 weeks. A different pattern was observed in tissue from frank tumors. Compared with normal-appearing mucosa, both sialomucin staining and tissue sialic acid levels were reduced in tumor tissue by 32 weeks. These studies indicated that tissue sialic acid levels may provide a simple and reliable screening technique in the early diagnosis of premalignant change in all regions of the colon.

Somatic mutation screening: identification of individuals harboring K-ras mutations with the use of plasma DNA.

BACKGROUND: Many cancers are attributed to somatic mutation of DNA. We investigated whether it is feasible to detect cancer-associated somatic mutations in patients with neoplasms by using plasma DNA. METHODS: Plasma samples were prospectively collected from 240 patients undergoing colonoscopy. Colorectal biopsies were performed as clinically indicated in 135 patients, and risk factor information was available from 232 patients. DNA was extracted from plasma and colorectal tissue and was amplified by use of a polymerase chain reaction method that enriches for mutations in codon 12 of the K-ras oncogene. Molecular, histologic, and clinical data were compared by use of two-sided Fisher's exact test. RESULTS: Mutations in the K-ras gene detected in the plasma of 64 (28%) of 232 patients were statistically significantly associated with colorectal cancer risk factors (P =.0002). Of those patients having tissue available for comparison (n = 135), mutations in the K-ras gene were found in the tissues of 35 patients, and 29 (83%) of these 35 showed mutations in plasma samples. In contrast, the plasma assay was negative in 93 of the 100 patients whose tissue K-ras was wild-type. Among patients without biopsies (n = 105), 28 had mutated K-ras in their plasma DNA, despite the absence of remarkable colonoscopy findings; 24 of these 28 patients had risk factors for colorectal cancer. Overall, 25 (39%) of 64 patients showing mutations in plasma DNA had colorectal neoplasms with K-ras mutations compared with five (3%) of 176 patients without K-ras mutations in plasma DNA. CONCLUSION: Plasma DNA assays for the detection of mutations in K-ras codon 12 may provide a feasible method to screen populations for somatic mutations frequently found in neoplasms. The clinical utility of using this test in screening populations requires further study.

Fine mapping of a genetic locus for Peutz-Jeghers syndrome on chromosome 19p.

Peutz-Jeghers syndrome (PJS) was recently mapped in a single report to the telomeric region of chromosome 19p (A. Hemminki et al., Nat. Genet., 15: 87-90, 1997). Our studies confirm this location and provide further localization of the PJS locus. In the five families examined, there were no recombinants with the marker D19S886. The multipoint log odds score at D19S886 is 7.52, and we found no evidence for genetic heterogeneity. We also found that all carriers expressed the PJS phenotype and no noncarriers displayed PJS sequellae, indicating complete penetrance with no sporadic cases.

Genotype-phenotype correlations in Peutz-Jeghers syndrome.

BACKGROUND AND AIMS: Peutz-Jeghers syndrome (PJS) is a dominantly inherited disorder often caused by mutations in STK11. Time to onset of symptoms was characterised for a large collection of individuals with PJS who had been tested for STK11 mutations and genotype-phenotype correlations were evaluated. METHODS: We characterised mutations in 42 independent probands and also used a historical cohort design to study 51 individuals with Peutz-Jeghers syndrome who had completed self-administered questionnaires. RESULTS: Mutations were detected in 22/32 (69%) probands with PJS and 0/10 probands referred to rule out PJS. Real-time PCR analysis to quantitate DNA failed to detect any large deletions in PJS participants without STK11 mutations. The median time to onset for gastrointestinal symptoms or polypectomy was 13 years of age but showed a wide variability. Gastric polyps were frequent in PJS participants, with a median age at onset of 16 years. Individuals with missense mutations had a significantly later time to onset of first polypectomy (p = 0.04) and of other symptoms compared with those participants either with truncating mutations or no detectable mutation. CONCLUSION: STK11 mutation analysis should be restricted to individuals who meet PJS criteria or their close relatives. Direct sequencing of STK11 yields a high rate of point mutations in individuals who meet phenotypic PJS criteria. Individuals with missense mutations of STK11 typically had a later time to onset for PJS symptoms. The common occurrence of gastric polyps may facilitate chemopreventive studies for this disorder.

Results of a television-advertised public screening program for colorectal cancer.

We report the results of a free, television-advertised mass screening program for colorectal cancer using stool guaiac kits. A total of 57,000 test kits were picked up and 29,619 (53%) were returned; 3.9% (1165) of the tests were positive. Ninety-three percent of persons with a positive screen sought medical evaluation after screening. Detailed follow-up was available on 744 persons. Fifty-eight persons had large-bowel carcinomas diagnosed, 80% of which were localized. One hundred sixty persons had adenomatous polyps removed. Forty percent of cancers and 58% of polyps were detected in persons with only one or two positive test slides out of a total of six. In 33% of persons with a positive screen, the diagnostic workup consisted of a repeated stool guaiac test and/or sigmoidoscopy only. A major drawback to improving the results of mass screening programs for colorectal cancer is the limited gastrointestinal workup conducted by physicians in many persons with a positive fecal occult blood test.

Apoptosis associated with esophageal adenocarcinoma: influence of photodynamic therapy.

Tumor cell death in vitro by photodynamic therapy (PDT) has been related to the induction of apoptosis. We measured and compared changes in apoptosis and caspase 3 activity, an effector of apoptosis, in normal and neoplastic esophageal tissues during PDT. Apoptosis index, caspase 3 cleavage activity, pro-caspase 3, p53, and bcl-2 levels were measured in normal and neoplastic tissues of patients with esophageal adenocarcinoma before, during, and after PDT with Photofrin. The apoptotic index was greater in carcinoma tissue compared to adjacent normal tissues. In concert, pro-caspase 3 immunoreactivity was absent and caspase 3-like cleavage activity was over 30-fold greater in carcinoma tissue compared to normal esophageal tissues. These parameters were unaffected by PDT. Variable changes in bcl-2 and p53 immunoreactivity were noted in normal and carcinoma tissues during PDT. Greater levels of apoptosis and caspase 3 activity are hallmarks of esophageal adenocarcinoma compared to normal esophageal tissue. These differences were unaffected by PDT. This may be due to the fact that tissues were obtained 72 h post-PDT therapy. Changes in these parameters may have occurred early after PDT therapy. An assessment of apoptosis and caspase 3 activity prior to 72 h post-PDT may provide further insight into the mechanism involved, although no sustained effects on these parameters by PDT were noted.

p53 and APC mutations are detectable in the plasma and serum of patients with colorectal cancer (CRC) or adenomas.

BACKGROUND: A variety of tumor-related DNA has been detected in the plasma and serum of cancer patients, including RAS, the BCL2-IgH transgene, and p53. It is not known whether the APC gene, which is frequently mutated in colorectal cancer (CRC), can be identified in the plasma of CRC patients. Similarly, it is unknown whether another tumor suppressor gene altered in CRC, p53, is detectable in people with precursor lesions to CRC. MATERIALS AND METHODS: The plasma of 240 colonoscopy patients was tested for mutations at two frequently altered sites (codons 175 and 248). A restriction enzyme-mediated enrichment assay was used to detect these mutants. We also tested tumor tissue from 8 patients with CRC for mutations in the mutation cluster region of the APC gene using direct DNA sequencing. Corresponding plasma from any positive patient was then similarly sequenced. RESULTS: Three plasma p53 mutations were identified. Two of these patients had adenomas at biopsy, and 1 had a hyperplastic polyp. All were tested for the same p53 mutations, and 1 of the adenomas was positive. One of the 8 CRC patients had a 5-base-pair deletion in the cancer and the same deletion was detectable in that patient's plasma, although at an amount that we estimate at 1-5% of the total APC DNA present. CONCLUSIONS: APC mutations are detectable in the plasma of CRC patients. p53 mutants are detectable in the plasma of colorectal adenoma patients. These may have important implications for cancer screening and diagnosis in the large intestine and suggest that all malignant and premalignant DNA alterations from the colon are identifiable in the blood.

Afferent loop obstruction presenting as acute pancreatitis and pseudocyst: case reports and review of the literature.

Afferent loop obstruction after gastrectomy and Billroth II reconstruction is an uncommon problem. Complete acute obstruction requires emergent laparotomy. However, chronic obstruction may begin insidiously and its symptoms may reflect other gastrointestinal diseases. Two patients are described who developed acute abdominal pain, marked hyperamylasemia, and palpable abdominal masses 5 and 15 years after Billroth II gastrectomy. The masses were initially interpreted as pancreatic pseudocysts. Both patients were found to have chronically obstructed afferent limbs, and in one the obstruction was associated with hundreds of stasis stones within the afferent limb. Surgical decompression was accomplished in each patient. Patients who have undergone Billroth II reconstruction have signs, symptoms, and laboratory findings consistent with acute pancreatitis. A history of previous gastrectomy, recurrent or severe abdominal pain, hyperamylasemia with characteristic tomography, and endoscopic findings will establish the diagnosis and necessitate surgical evaluation and intervention.

Effect of selenium on growth, S-adenosylmethionine and polyamine biosynthesis in human colon cancer cells.

Selenium metabolism and polyamine biosynthesis are linked in their common requirement for S-adenosylmethionine. The effects of selenium supplementation (0.1 to 6.0 ppm) on growth, polyamine biosynthesis and S-adenosylmethionine were examined in two human colon cancer cell lines, WiDr and HT29. WiDr cells were very sensitive to selenium with a significant decrease in 3H-thymidine incorporation and cell number to doses above 0.25 ppm. HT29 cells were less sensitive. In HT29 cells, ornithine decarboxylase activity and its product putrescine decreased in parallel with the growth inhibitory effects of selenium. Similar changes were not noted in WiDr cells. Spermidine and spermine content were conserved in both cell lines exposed to cytotoxic doses of selenium. S-adenosylmethionine was increased in HT29 cells at cytotoxic doses of selenium (1.0 to 6.0 ppm).

Peutz-Jeghers syndrome: clinicopathology and molecular alterations.

Peutz-Jeghers syndrome (PJS, OMIM 175200) is an unusual inherited intestinal polyposis syndrome associated with distinct peri-oral blue/black freckling. Variable penetrance and clinical heterogeneity make it difficult to determine the exact frequency of PJS. PJS is a cancer predisposition syndrome. Affected individuals are at high risk for intestinal and extra-intestinal cancers. In 1997, linkage studies mapped PJS to chromosome 19p, and subsequently a serine/threonine kinase gene defect (LKB1) was noted in a majority of PJS cases. A phenotypically similar syndrome has been produced in an LKB1 mouse knockout model. Several PJS kindred without LKB1 mutations have been described, suggesting other PJS loci. The management of PJS is complex and evolving. New endoscopic technologies may improve management of intestinal polyposis. Identification of specific genetic mutations and their targets will more accurately assess the clinical course, and help gage the magnitude of cancer risk for affected individuals.

Selenium and difluoromethylornithine additively inhibit DMH-induced distal colon tumor formation in rats fed a fiber-free diet.

We investigated the effects of difluoromethylornithine, an inhibitor of ornithine decarboxylase (ODC) and selenium supplementation on tumor formation induced by the carcinogen 1,2-dimethylhydrazine (DMH) in Sprague-Dawley rats. A biochemical link between polyamine biosynthesis and selenium metabolism to its cancer preventative form has been suggested by the common requirement of S-adenosylmethionine. One-hundred and twenty male Sprague-Dawley rats were divided into experimental (n = 80) and control (n = 40) groups. Experimental animals received DMH 20 mg/kg s.c. for 20 weeks. Animals were fed either a regular diet (selenium content 0.2 p.p.m.) or a high selenium diet (5 p.p.m.) with or without 0.2% DFMO in the drinking water. At death, week 30, animal weights within experimental or control groups were not different between the four diet treatment groups. Tumor number and incidence in the proximal colon was not affected by DFMO treatment, selenium supplementation or the combined treatment. In contrast, in the distal colon, 19 tumors developed in the DFMO treated group, 22 tumors in the high selenium group and only 12 tumors in the combined high selenium/DFMO treatment group compared to 32 tumors in the regular diet group. Similarly, tumor incidence was decreased by DFMO and selenium supplementation and their effects were additive. In control animals, ODC activity was decreased by DFMO treatment and selenium supplementation in the distal colon and liver, but not the proximal colon. ODC activity of tumor tissue was greater than normal colon tissue from diet paired animals for proximal and distal colon, except for distal colonic tumors in the high selenium/DFMO treatment group. Polyamine content, however, did not correlate with ODC activity in normal or neoplastic tissue. In general, S-adenosylmethionine levels from normal colon and liver tissue were unaffected by diet treatment. Selenium supplementation in combination with DFMO treatment selectively inhibited distal colon tumor formation in rats fed a fiber-free diet.

Treatment strategies for Helicobacter pylori infection.

Peptic ulcer disease is strongly associated with infection by Helicobacter pylori, a spiral-shaped, flagellated organism found predominantly in the gastric antrum. More than 90 percent of duodenal ulcers and adenocarcinomas of the distal stomach are associated with H. pylori infection. Eradication of the organism effectively prevents relapses of gastroduodenal ulcers associated with H. pylori. In patients undergoing endoscopy, the rapid urease test is highly sensitive and specific in diagnosing H. pylori infection. Noninvasive diagnostic methods include serologic antibody measurements and urea breath testing. Empiric therapy may be tried if the diagnosis is suspected on a clinical basis. Traditional 14-day "triple therapy" with bismuth, metronidazole and either amoxicillin or tetracycline has consistently produced eradication rates of approximately 90 percent. Newer combination regimens have shown promise in a smaller number of studies. No single agent given as monotherapy has proved to be acceptably effective in clinical studies.

Protein kinase C activity as a potential marker for colorectal neoplasia.

Protein kinase C (PKC) is a mediator of transmembrane signal transduction, important in cell growth and differentiation. Cell activation by extracellular signals is associated with a translocation of PKC from the cytosol to the membrane. We measured and compared PKC activity in cytosol and membrane fractions of normal and neoplastic colorectal tissue. Total and membrane-associated PKC activity in normal colorectal tissue was greater in patients (N = 16) with colorectal cancer compared to that from patients with a normal colonoscopy (N = 16), P < 0.01. A similar trend was noted in PKC activity of normal colorectal tissue from patients with adenomas compared to patients with a normal colonoscopy. PKC activity (total, membrane-associated, percent membrane) was not different in neoplastic colorectal tissue compared to that of adjacent normal tissue. However, there was a considerable range of PKC activity noted in all groups, which would limit the utility of PKC activity as a marker for colorectal neoplasia.

Interspecies comparison of calmodulin binding proteins throughout the gastrointestinal tract: comparison with human colon adenomas and adenocarcinomas.

Calmodulin is an ubiquitous cytoplasmic protein which mediates many of the actions of calcium on intestinal tissue including regulation of growth and differentiation of normal and neoplastic cells. Using a biotinylated calmodulin overlay system, we compared the pattern of calmodulin binding proteins throughout the gastrointestinal tract of mice, rats, rabbits, and humans, and in human colonic adenomas and adenocarcinomas. A common calmodulin binding protein of 67 kDa was found in membrane and cytosolic fractions of oesophagus, stomach, proximal and distal small intestine, and colon from all four species. In human tissue this 67 kDa protein was present in greatest concentration in stomach tissue. Furthermore, a 67 kDa binding protein was the major calmodulin binding protein from human stomach and ileum as determined by ion exchange and calmodulin affinity chromatography. A similar pattern of binding proteins was noted between rabbit and human cytosolic fractions; proteins of 60/67 kDa and 105 kDa were present in stomach tissue. A 94 kDa protein was present in samples of rabbit and human ileum but not of mouse or rat. A similar pattern of calmodulin binding proteins was seen in normal and neoplastic large bowel tissue, apart from one of nine adenocarcinomas, where a distinct 54 kDa band was noted in both cytosolic and membrane fractions. The results of this study show interspecies and organ differences between calmodulin binding proteins, but suggest that a 67 kDa protein is the major binding protein present throughout normal gastro-intestinal tract and neoplastic human tissue.

Alterations in lectin binding in the proximal and distal colon of Sprague-Dawley rats with 1,2 dimethylhydrazine administration.

Sequential changes in rhodamine or fluorescein isothiocyanate-conjugated lectin binding of proximal and distal colonic crypts were studied during and after the administration of the 1,2 dimethylhydrazine (DMH). Five adult its unexposed to DMH or vehicle served as baseline controls. Tissue from normal appearing colon and tumor tissue was incubated with Ulex europaeus agglutinin 1 (UEA), Arachis hypogaea (PNA), Dolichos biflorus (DBA) and Griffonia simplicafolia 1 (GSA1). Distinct regional differences were noted in the baseline controls. UEA, PNA and DBA binding were absent in the distal colonic crypt cells. In the proximal colon minimal UEA and PNA binding was noted in the lower crypt whereas DBA binding was intense. GSA1 binding was diffuse in the upper and lower crypt of both regions. During carcinogenesis a progressive increase in PNA binding was noted in normal appearing colonic crypts from both regions. A progressive increase in PNA binding in proximal and distal colonic tumors was noted over time. Similar to normal tissue, DBA bound markedly to proximal colon tumors but was absent in most distal colonic tumors. UEA stained all proximal tumors intensely at all time points. In distal colonic tumors, UEA staining was diminished at 30 weeks compared to tumors analyzed at 16, 22 and 26 weeks. Mucin depletion was also a feature of tumor tissue compared to adjacent normal and hyperplastic glands. This study documents the region specific changes in lectin binding in normal and neoplastic colonic crypts induced by DMH.

Cellular proliferation in proximal and distal rat colon during 1,2-dimethylhydrazine-induced carcinogenesis.

Sequential changes in proliferative parameters in proximal and distal colonic crypts were studied during 1,2-dimethylhydrazine-induced carcinogenesis using [3H]thymidine autoradiography as a probe. 1,2-dimethylhydrazine (20 mg/kg) and vehicle (ethylenediaminetetraacetic acid) control rats received weekly s.c. injections for 20 wk. All animals received a pulse of [3H]thymidine before death at weeks 2, 6, 10, 16, 22, 26, or 30. In addition, 8 animals unexposed to 1,2-dimethylhydrazine or vehicle served as baseline controls. Dramatic regional differences were noted in the baseline controls. Crypt length, labeling index, and proliferative zone size were all significantly greater distally than proximally (p less than 0.05), whereas the labeling index of the proliferative zone tended to be enhanced proximally. During 1,2-dimethylhydrazine treatment the crypt length, labeling index, and proliferative zone size increased in both regions. As these parameters changed in parallel, the differences between proximal and distal colon did not change significantly during carcinogenesis. Actual tumor formation did differ, however, with tumors appearing earlier and in greater abundance in the distal colon. These findings show similar proliferative changes in both the proximal and distal colon during 1,2-dimethylhydrazine treatment and indicate that the enhanced baseline proliferative state of the distal colon compared with the proximal colon must be considered in the process of tumor formation.

Lectin histochemistry of adenomatous polyps. Not a predictor of metachronous lesions.

The binding characteristics of fluorescein isothiocyanate conjugated lectins to normal colonic mucosa, and 43 adenomatous polyps were studied by fluorescence microscopy. The lectin, Dolichos biflorus agglutinin (DBA) stained intensely to upper crypt cells of the sigmoid colon and rectum but to a lesser degree to proximal colonic crypts or lower crypt cells distally. Peanut agglutinin (PNA) and Ulex europaeus agglutinin (UEA) did not bind to the theca of proximal or distal colonic crypts. The lectin Griffonia simplicafolia agglutinin (GSA1) bound intensely to upper and lower crypt cells of both regions. PNA binding was noted in 56% of adenomatous polyps, occurred more often in polyps of the distal colorectum, and increased with polyp size and villous histology. UEA bound to 26% of adenomatous polyps, 42% of proximal polyps, and 17% of distal polyps. DBA staining was noted in 72% of polyps without regional preference. GSA1 stained all polyp specimens. To determine if the lectin binding characteristics of an index (initial) polyp might serve as a predictor of metachronous lesions, 20 patients (29 polyps) without a history of polyps or cancer and who had at least one surveillance colonoscopy 1 to 3 years after the initial polypectomy were studied. The presence or absence of PNA, UEA, or DBA binding in an index polyp did not predict the occurrence of metachronous lesions. Five of the six patients with more than one index polyp had metachronous polyps at follow-up surveillance colonoscopy.

Peutz-Jeghers syndrome: a clinicopathologic survey of the "Harrisburg family" with a 49-year follow-up.

Of the original Peutz-Jeghers families reported by Jeghers, the "Harrisburg Family" has now been followed for 49 yr. Their 12 affected family members comprise the largest Peutz-Jeghers kindred reported. The course of this family illustrates that Peutz-Jeghers syndrome is not a benign disease. One family member developed a duodenal carcinoma in a hamartoma with adenomatous changes; this progression in the duodenum has not previously been reported. Ten patients underwent 75 polypectomies. One patient developed short bowel syndrome. Three patients died in young adulthood. The development of gastrointestinal malignancy in 2 of 12 affected patients suggests that Peutz-Jeghers syndrome may be a premalignant condition. Consequently, even asymptomatic gastric, duodenal, and colonic polyps should be removed endoscopically. If surgical intervention is necessary, intraoperative endoscopy with polypectomy may prevent the development of a short bowel syndrome. Colonoscopic screening of patients and their family members may be beneficial and surveillance for extraintestinal malignancy appears to be warranted.